Human DNA Ligase III

ABSTRACT

A human DNA Ligase III polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide via gene therapy for the treatment of disorders associated with a defect in DNA Ligase III. Antagonists against such polypeptides and their use as a therapeutic to destroy unwanted cells are also disclosed. Diagnostic assays to detect mutant DNA Ligase III genes are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/879,228, filed Jun. 13, 2001, which is a divisional of U.S.application Ser. No. 09/054,775, filed Apr. 3, 1998, (now U.S. Pat. No.issued 6,284,504, issued Sep. 4, 2001), which is a divisional of U.S.application Ser. No. 08/464,402, filed Jun. 5, 1995 (now U.S. Pat. No.5,858,705, issued Jan. 12, 1999), which is a continuation-in-part ofInternational Application No. PCT/US95/03939, filed Mar. 31, 1995. Eachof the above cited patents and patent applications are incorporated byreference herein.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. The polypeptide of the presentinvention has been putatively identified as Human DNA Ligase III. Theinvention also relates to inhibiting the action of such polypeptides.

BACKGROUND OF THE INVENTION

[0003] DNA strand breaks and gaps are generated transiently duringreplication, repair and recombination. In mammalian cell nuclei,rejoining of such strand breaks depends on several different DNApolymerases and DNA ligase enzymes.

[0004] The mechanism for joining of DNA strand interruptions by DNAligase enzymes has been widely described. The reaction is initiated bythe formation of a covalent enzyme-adenylate complex. Mammalian andviral DNA ligase enzymes employ ATP as cofactor, whereas bacterial DNAligase enzymes use NAD to generate the adenylyl group. The ATP iscleaved to AMP and pyrophosphate with the adenylyl residue linked by aphosphoramidate bond to the ε-amino group of a specific lysine residueat the active site of the protein (Gumport, R. I., et al., PNAS,68:2559-63 (1971)). Reactivated AMP residue of the DNA ligase-adenylateintermediate is transferred to the 5′ phosphate terminus of a singlestrand break in double stranded DNA to generate a covalent DNA-AMPcomplex with a 5′-5′ phosphoanhydride bond. This reaction intermediatehas also been isolated for microbial and mammalian DNA ligase enzymes,but is more short lived than the adenylylated enzyme. In the final stepof DNA ligation, unadenylylated DNA ligase enzymes required for thegeneration of a phosphodiester bond catalyze displacement of the AMPresidue through attack by the adjacent 3′-hydroxyl group on theadenylylated site.

[0005] The occurrence of three different DNA ligase enzymes, DNA LigaseI, II and III, was established previously by biochemical andimmunological characterization of purified enzymes (Tomkinson, A. E. etal., J. Biol. Chem., 266:21728-21735 (1991) and Roberts, E., et al., J.Biol. Chem., 269:3789-3792 (1994)). However, the inter-relationshipbetween these proteins was unclear as a cDNA clone has only beenavailable for DNA Ligase I, the major enzyme of this type inproliferating cells (Barnes, D. E., et al, PNAS USA, 87:6679-6683(1990)). The main function of DNA Ligase I appears to be the joining ofOkazaki fragments during lagging-strand DNA replication (Waga, S., etal., J. Biol. Chem. 269:10923-10934 (1994); Li, C., et al., Nucl. AcidsRes., 22:632-638 (1994); and Prigent, C., et al., Mol. Cell. Biol.,14:310-317 (1994)).

[0006] A full-length human cDNA encoding DNA Ligase I has been obtainedby functional complementation of a S. cereviasiae cdc9temperature-sensitive DNA ligase mutant (Barker, D. G., Eur. J.Biochem., 162:659-67 (1987)). The full-length cDNA encodes a 102-kDaprotein of 919 amino acid residues. There is no marked sequence homologyto other known proteins except for microbial DNA ligase enzymes. Theactive site lysine residue is located at position 568. It alsoeffectively seals single-strand breaks in DNA and joins restrictionenzyme DNA fragments with staggered ends. The enzyme is also able tocatalyze blunt-end joining of DNA. DNA Ligase I can join oligo (dT)molecules hydrogen-bonded to poly (dA), but the enzyme differs from T4DNA Ligase II and III in being unable to ligate oligo (dT) with a poly(rA) complementary strand.

[0007] Human DNA Ligase III is more firmly associated with the cellnuclei. This enzyme is a labile protein, which is rapidly inactivated at42° C. DNA Ligase III resembles other eukaryotic DNA Ligase enzymes inrequiring ATP as cofactor, but the enzyme differs from DNA Ligase I inhaving a higher association for ATP. DNA Ligase III catalyzes theformation of phosphodiester bonds with an oligo (dT) poly (rA)substrate, but not with an oligo (rA) poly (dT) substrate, so it differscompletely from DNA Ligase I in this regard (Arrand, J. E. et al., J.Biol. Chem., 261:9079-82 (1986)).

[0008] DNA Ligase III repairs single strand breaks in DNA efficiently,but it is unable to perform either blunt-end joining or AMP-dependentrelaxation of super-coiled DNA (Elder, R. H. et al., Eur. J. Biochem.,203:53-58 (1992)).

[0009] Clues as to the physiological role of DNA Ligase III have comefrom its physical interaction in a high salt-resistant complex withanother nuclear protein, the XRCC1 gene product (Caldecott, K. W., etal., Mol. Cell. Biol., 14:68-76 (1994) and Ljungquist, S., et al.,Mutat. Res., 314:177-186 (1994)). The XRCC1 gene encodes a 70 kDaprotein, that by itself does not appear to join DNA strand breaks(Caldecott, K. W., et al., Mol. Cell. Biol. 14:68-76 (1994); Ljungquist,S., et al., Mutat. Res., 314:177-186 (1994) and Thompson, L. H., et al.,Mol. Cell. Biol., 10:6160-6171 (1990)). However, mutant rodent cellsdeficient in XRCC1 protein exhibit reduced DNA Ligase III activity,defective strand break repair, an anomalously high level of sisterchromatid exchanges, are hyper-sensitive to simple alkylating agents andionizing radiation, and have an altered mutation spectrum after exposureto ethyl methanesulfonate (Caldecott, K. W., et al., Mol. Cell. Biol.,14:68-76 (1994); Ljungquist, S., et al., Mutat. Res., 314:177-186(1994); Thompson, L. H., et al., Mol. Cell. Biol., 10:6160-6171 (1990);and Op het Veld, C. W., et al., Cancer Res., 54:3001-3006 (1994)). Thesedata indicate that XRCC1 mutant cells are defective in baseexcision-repair, and strongly suggest that both DNA Ligase III and XRCC1are active in this process (Dianov, G., and Lindahl, T., Curr. Biol.,4:1069-1076 (1994)).

[0010] A purified mammalian protein fraction active in repair andrecombination processes in vitro was shown to contain a ligase with theproperties of Human DNA Ligase III, but no detectable amounts of HumanDNA Ligase I (Jessberger, R., et al., J. Biol. Chem., 268:15070-15079(1993)). The role of the distinct enzyme, DNA Ligase II, remainsunclear, although an observed increase in DNA Ligase II activity duringmeiotic prophase suggests a role in meiotic recombination (Higashitani,A., et al., Cell Struct. Funct., 15:67-72 (1990)). Comparison of³²P-adenylylated DNA Ligase II and III by partial or completeproteolytic cleavage patterns indicated that these two enzymes shareextensive amino acid sequence similarity or identity flanking theiractive sites, but that they are quite different from DNA Ligase I(Roberts, E., et al., J. Biol. Chem., 269:3789-3792 (1994)). Neither DNALigase I, II nor III is exclusively a mitochondrial enzyme.

BRIEF SUMMARY OF THE INVENTION

[0011] The polynucleotide of the present invention and polypeptideencoded thereby have been putatively identified as human DNA Ligase IIIas a result of size, amino acid sequence homology to DNA Ligase II andability to bind XRCC1 protein. Heretofore, the gene sequence of DNALigase III was not known.

[0012] In accordance with one aspect of the present invention, there areprovided novel mature polypeptides which are human DNA Ligase III, aswell as biologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof.

[0013] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding human DNA LigaseIII, including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs andbiologically active and diagnostically or therapeutically usefulfragments thereof.

[0014] In accordance with yet a further aspect of the present invention,there is provided a process for producing such polypeptides byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing a human DNA Ligase III nucleicacid sequence, under conditions promoting expression of said protein andsubsequent recovery of said protein.

[0015] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

[0016] In accordance with another aspect of the present invention thereis provided a method of treating conditions which are related toinsufficient human DNA Ligase III activity via gene therapy comprisinginserting the DNA Ligase III gene into a patient's cells either in vivoor ex vivo. The gene is expressed in transduced cells and as a result,the protein encoded by the gene may be used therapeutically, forexample, to prevent disorders associated with defects in DNA, forexample, abnormal cellular proliferation, for example cancers, leukemiaand tumors, to treat severe immunosuppression, stunted growth andlymphoma, as well as cellular hypersensitivity to DNA-damaging agents.

[0017] In accordance with yet a further aspect of the present invention,there is also provided nucleic acid probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to human DNALigase III sequences which may be used diagnostically to detect amutation in the gene encoding DNA Ligase III.

[0018] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides, which may bemanufactured intracellularly or administered through gene therapy forinhibiting the action of such polypeptides, for example, to target anddestroy undesired cells, e.g., cancer cells.

[0019] In accordance with still another aspect of the present invention,there are provided diagnostic assays for detecting mutations in thepolynucleotide sequences of the present invention for detecting diseasesrelated to a lack of Human DNA Ligase III activity.

[0020] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0022] FIGS. 1A-1J show the cDNA sequence (SEQ ID NO:1) and thecorresponding deduced amino sequence (SEQ ID NO:2) of the DNA Ligase IIIpolypeptide. The standard one letter abbreviation for amino acids isused. The vertical arrow indicates the active site lysine.

DETAILED DESCRIPTION

[0023] In accordance with an aspect of the present invention, there isprovided an isolated nucleic acid (polynucleotide) (SEQ ID NO:1) whichencodes for the mature polypeptide having the deduced amino acidsequence of FIGS. 1A-1J (SEQ ID NO:2) or for the mature polypeptideencoded by the cDNA of the clone deposited as ATCC Deposit No. 97052 onFeb. 6, 1995 with the American Tissue Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209, USA. The strain is beingmaintained under the terms of the Budapest Treaty and will be madeavailable to a patent office signatory to the Budapest Treaty.

[0024] A polynucleotide encoding a polypeptide of the present inventionmay be obtained from testis, prostate, heart and thymus. Thepolynucleotide of this invention was discovered in a cDNA libraryderived from human testis. It is structurally related to the DNA ligasefamily. It contains an open reading frame encoding a protein of 922amino acid residues. The protein exhibits the highest degree of homologyto vaccine virus DNA ligase with 56% identity and 73% similarity overthe entire protein. It is also important that there is a conservedactive lysine residue at position 421 which is bordered on either sideby a hydrophobic amino acid residue, and the sequence E-KYDG-R (SEQ IDNO:11) is also conserved and is common to enzymes from different sourcessuch as mammalian cells, yeasts, vaccinia virus and bacteriophage T7.

[0025] The region flanking the conserved lysine residue is an activesite motif that is essential for the formation of an enzyme-adenylatereaction intermediate (Tomkinson, A. E., et al., PNAS USA, 88:400-404(1991)). The conserved lysine residue is indicated by a vertical arrowand the active site motif is underlined in FIGS. 1A-1J. Further aputative zinc finger motif shown at residues 18 to 55 in FIGS. 1A-1J isunderlined by a broken line. The 100 kDa in vitro translation product ofthe DNA ligase III cDNA interacts with human XRCC1 protein which is acharacteristic of DNA Ligase III (Caldecott, K. W., et al., Mol. Cell.Biol., 14:68-76 (1994)). Histidine-tagged recombinant XRCC1 protein wasincubated with [³⁵S] methionine-labeled in vitro translation product ofthe cDNA to allow formation of XRCC1-protein complexes, after whichNTA-agarose beads were added to affinity-bind XRCC1-His. The agarosebeads were washed to remove non-specifically associated polypeptidesprior to elution of XRCC1-His with 200 mM imidazole. XRCC1-his binds theproduct of the cDNA. Recovery of radiolabeled polypeptides is dependenton addition of XRCC1-His. Approximately 50% of the full length 100 kDatranslation product, and as much as 90% of some of the truncatedpolypeptides, were recovered with XRCC1-His. These results indicate thatthe cDNA clone encodes a 100 kDa polypeptide.

[0026] The longest open reading frame of the cDNA encoding DNA ligaseIII extends from 73 bp to 3099 bp within the cDNA clone and would encodea polypeptide of 1009 amino acids, approximately 150 kDa molecular mass.The next downstream ATG at 334 bp occurs in a typical translation startconsensus and defines an open reading frame of 2766 bp (922 aminoacids). The protein produced in this case would be approximately 103kDa, consistent with both the observed molecular mass of the in vitrotranslation product and the apparent molecular mass of authentic DNALigase III purified from HeLa cells by standard chromatographicprocedures. This indicates that this cDNA represents a full length cDNAclone. Furthermore, a 5′-truncated cDNA clone lacking the first 78 bp(and the first ATG codon) produced an in vitro translation product ofidentical electrophoretic mobility to that encoded by the full lengthclone, in support of assignment of the ATG at 334 bp as the translationinitiation codon.

[0027] The DNA Ligase III amino acid sequence shows extensive amino acidhomology to Human DNA Ligase I. The DNA Ligase III sequence is identicalat 8 of 12 residues flanking the active site lysine of DNA Ligase I, andboth contain the minimum active site consensus for all ATP-dependent DNAligases, —K-DG-R— (SEQ ID NO:10), with lys₄₂₁ (DNA Ligase III) being theputative active lysine. Although their amino acid sequences are notco-linear at optimum alignment, human DNA Ligase I and III differ by 9amino acids in the size of the region between the two motifs (activelysine and minimum active site motifs).

[0028] The 3′ flanking motif is located 37 amino acids from theC-terminus of DNA Ligase I, whereas the DNA Ligase III sequence extendsa further 1.95 residues. The C-terminus of the DNA Ligase III shows weakhomology to several proteins, including approximately 20% identity to a144 amino acid sequence within the C-terminal quarter of both human andmurine XRCC1.

[0029] In their N-terminal regions, DNA Ligase I and III show verylimited sequence homology beyond about 30 residues upstream of theiractive sites, and DNA Ligase I has an extended hydrophilic N-terminalregion with no homology to DNA Ligase.

[0030] The N-terminal 112 amino acids of the DNA Ligase III cDNA showapproximately 30% identity to residues 3 to 107, and also residues 108to 217, of human poly (ADP ribose) polymerase (PARP). These same tworegions contain two evolutionarily conserved zinc finger motifs withinthe DNA-binding domain of PARP.

[0031] The highly conserved motif flanking the 3′ boundary of the regionof homology between DNA Ligase I and III is unique to ATP-dependent DNAligases and is not found in the RNA capping enzymes. Similarly tovaccinia virus DNA Ligase, Human DNA Ligase III does not contain theregion 2 motif which is present in the capping enzymes, and Human DNALigase I (Shuman, S., et al. PNAS USA, in press (1994)).

[0032] There is near identity of peptides within the predicted aminoacid sequence of the DNA Ligase III cDNA with sequenced tryptic peptidesfrom the 70 kDa bovine DNA Ligase II protein (Wang, Y-C. J., et al., J.Biol. Chem., 269:31923-31928 (1994)). These tryptic peptides span theregion between the active site and the conserved DNA Ligase-specificmotif, and are also highly homologous to the corresponding region of thevaccinia virus DNA ligase. The sequence ₄₁′-(K) CPNGMFSEIKYDGERVQVH(K)-₄₃₁ (SEQ ID NO:9) in the predicted amino acid sequence of the DNAligase III cDNA, with Lys₄₂₁ the putative active lysine, is identical tothe active site tryptic peptide identified in the purified bovine DNALigase II protein and different from that of DNA Ligase I (Tomkinson, A.E., et al., PNAS USA, 88:400-404 (1991)).

[0033] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIGS. 1A-1J (SEQ ID NO:1) orthat of the deposited clone or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same mature polypeptide as the DNA of FIGS.1A-1J (SEQ ID NO:1) or the deposited cDNA.

[0034] The polynucleotide which encodes for the mature polypeptide ofFIGS. 1A-1J (SEQ ID NO:2) or for the mature polypeptide encoded by thedeposited cDNA may include: only the coding sequence for the maturepolypeptide; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature polypeptide.

[0035] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0036] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIGS. 1A-1J (SEQ ID NO:2) or the polypeptide encoded by thecDNA of the deposited clone. The variant of the polynucleotide may be anaturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

[0037] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIGS. 1A-1J (SEQ ID NO:2) or thesame mature polypeptide encoded by the cDNA of the deposited clone aswell as variants of such polynucleotides which variants encode for afragment, derivative or analog of the polypeptide of FIGS. 1A-1J (SEQ IDNO:2) or the polypeptide encoded by the cDNA of the deposited clone.Such nucleotide variants include deletion variants, substitutionvariants and addition or insertion variants.

[0038] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIGS. 1A-1J (SEQ ID NO:1) or of the coding sequence ofthe deposited clone. As known in the art, an allelic variant is analternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

[0039] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0040] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0041] Fragments of the full length gene of the present invention may beused as a hybridization probe for a cDNA library to isolate the fulllength cDNA and to isolate other cDNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 30 bases and may contain, for example, 50or more bases. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene including regulatory and promoterregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0042] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIGS. 1A-1J (SEQ ID NO:1)or the deposited cDNA(s).

[0043] Alternatively, the polynucleotide may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO:1, for example, for recoveryof the polynucleotide or as a diagnostic probe or as a PCR primer.

[0044] Thus, the present invention is directed to polynucleotides havingat least a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

[0045] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0046] The present invention further relates to a DNA Ligase IIIpolypeptide which has the deduced amino acid sequence of FIGS. 1A-1J(SEQ ID NO:2) or which has the amino acid sequence encoded by thedeposited cDNA, as well as fragments, analogs and derivatives of suchpolypeptide.

[0047] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIGS. 1A-1J (SEQ ID NO:2) or that encoded by thedeposited cDNA, means a polypeptide which retains essentially the samebiological function or activity as such polypeptide.

[0048] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

[0049] The fragment, derivative or analog of the polypeptide of FIGS.1A-1J (SEQ ID NO:2) or that encoded by the deposited cDNA may be (i) onein which one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, which is employed for purificationof the mature polypeptide. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein.

[0050] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0051] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0052] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0053] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and more preferably at least90% similarity (more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least 95%similarity (still more preferably at least 95% identity) to thepolypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0054] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0055] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0056] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0057] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the DNA Ligase III genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

[0058] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies.

[0059] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0060] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0061] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0062] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0063] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0064] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX1.74, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0065] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(P),P_(L), and trp. Eukaryotic promoters include CMV immediate early, HSVthymidine kinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0066] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0067] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0068] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0069] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0070] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation, initiation andtermination sequences. Optionally, the heterologous sequence can encodea fusion protein including an N-terminal identification peptideimparting desired characteristics, e.g., stabilization or simplifiedpurification of expressed recombinant product.

[0071] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0072] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0073] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0074] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0075] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well known to those-skilled in the art.

[0076] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0077] The DNA Ligase III polypeptide can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0078] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0079] The DNA Ligase III polypeptides and agonists and antagonistswhich are polypeptides, described below, may be employed in accordancewith the present invention by expression of such polypeptides in vivo,which is often referred to as “gene therapy.”

[0080] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

[0081] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

[0082] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0083] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0084] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0085] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, φ-2, φ-AM, PA12, T19-14X, VT-19-17-H2, φCRE, φCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0086] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0087] Once the DNA Ligase III polypeptide is being expressedintracellularly via gene therapy, it may be used to repair single-strandbreaks in DNA which result from DNA-damaging agents, e.g., UV radiation.Several human syndromes result from autosomal recessive inheritance forthe DNA ligase gene. These syndromes cause severe immunodeficiency andgreatly increases the susceptibility of abnormal cellulardifferentiation due to the disrepair of DNA while at the cellular levelthey are characterized by chromosome instability and hypersensitivity toDNA-damaging agents. These syndromes include Fanconi's anemia andBlackfan-diamond anemia.

[0088] The polypeptide of the present invention may also be employed totreat severe immunosuppression which is the result of a defect in theDNA Ligase III gene. DNA Ligase III may also be employed to treatstunted growth and lymphoma which result from defective rejoining ofDNA.

[0089] Chromosome abnormalities in the 17q11-12 region, to which the DNALigase III gene has been mapped, are associated with several diseasesincluding several neoplasias. The most common neoplastic chromosomalabnormality in this region is a translocation between chromosomes 15 and17 seen in acute myeloid leukemia subtype m3 which involves thedisruption of the retinoic acid receptor a gene (Chomienne, H., et al.,Nature, 347:558-561 (1990)). However, chromosomal abnormalities in thisregion are frequently reported in both acute myeloid and lymphoblasticleukemias and are seen sporadically in several other cancers (Mitelman,F., Catalog of Chromosome Aberrations in Cancer (Fourth Edition), WileyLiss, New York (1991)). Accordingly, the DNA Ligase III gene and geneproduct may be employed to treat these neoplasias.

[0090] Fragments of the full length Ligase III gene may be used as ahybridization probe for a cDNA library to isolate other genes which havea high sequence similarity to the DNA Ligase III gene or have similarbiological activity. Probes of this type have at least 20 bases.Preferably, however, the probes have at least 30 bases and may contain,for example, 50 or more bases. The probe may also be used to identify acDNA clone corresponding to a full length transcript and a genomic cloneor clones that contain the complete DNA Ligase III gene includingregulatory and promoter regions, exons, and introns.

[0091] An example of a screen comprises isolating the coding region ofthe DNA Ligase III gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

[0092] The polypeptide and/or polynucleotide of the present inventionmay also be employed in relation to scientific research, synthesis ofDNA and for the manufacture of DNA vectors. The polypeptide and/orpolynucleotide of the present invention may be sold into the researchmarket. Thus, for example DNA Ligase III may be used for ligation of DNAsequences in vitro in a manner similar to other DNA ligase enzymes ofthe art.

[0093] This invention also provides a method of screening compounds toidentify those which enhance or inhibit the DNA-joining reactioncatalyzed by human DNA Ligase III. An example of such a method comprisescombining ATP, DNA Ligase III and DNA having single-strand breaks withthe compound under conditions where the DNA Ligase would normally cleaveATP to AMP and the AMP is transferred to the 5′ phosphate terminus of asingle strand break in double-stranded DNA to generate a covalentDNA-AMP complex with the single strand break being subsequentlyrepaired. The DNA having the single-strand breaks may be supplied in theabove example by mutant cells which are deficient in proteins that areresponsible for strand break repair, for example, mutant rodent cellsdeficient in XRCC1 and the cdc9 S. Cerevisiae DNA ligase mutant. Theability of the compound to enhance or block the catalysis of thisreaction could then be measured to determine if the compound is aneffective agonist or antagonist.

[0094] Human DNA Ligase III is produced and functions intracellularly,therefore, any antagonist must be intra-cellular. Potential antagoniststo human DNA Ligase III include antibodies which are producedintracellularly. For example, an antibody identified as antagonizing DNALigase III may be produced intracellularly as a single chain antibody byprocedures known in the art, such as transforming the appropriate cellswith DNA encoding the single chain antibody to prevent the function ofhuman DNA Ligase III.

[0095] Another potential antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5′ coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al,Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),thereby preventing transcription and the production of DNA Ligase III.The antisense RNA oligonucleotide hybridizes to the mRNA in vivo andblocks translation of the mRNA molecule into the DNA Ligase III(antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of DNA Ligase III.

[0096] Yet another potential antagonist includes a mutated form, ormutein, of DNA Ligase III which recognizes DNA but does not repairsingle-strand breaks and, therefore, acts to prevent human DNA LigaseIII from functioning.

[0097] The antagonists may be employed to target undesired cells, e.g.,cancer cells and leukemic cells, since the prevention of DNA Ligase IIIprevents repair of single-strand breaks in DNA and will eventuallyresult in death of the cell.

[0098] The small molecule agonists and antagonists of the presentinvention may be employed in combination with a suitable pharmaceuticalcarrier. Such compositions comprise a therapeutically effective amountof the molecule and a pharmaceutically acceptable carrier or excipient.Such a carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration.

[0099] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the pharmaceutical compositions of the present invention maybe employed in conjunction with other therapeutic compounds.

[0100] The pharmaceutical compositions may be administered in aconvenient manner such as by the oral, topical, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.

[0101] This invention also provides the use of the human DNA Ligase IIIgene as a diagnostic. For example, some diseases result from inheriteddefective genes. These genes can be detected by comparing the sequenceof the defective gene with that of a normal one. That is, a mutant genewould be associated with hypersensitivity to DNA-damaging agents and anelevated susceptibility to abnormal cell growth, for example, tumors,leukemia and cancer.

[0102] Individuals carrying mutations in the human DNA Ligase III genemay be detected at the DNA level by a variety of techniques. Nucleicacids used for diagnosis may be obtained from a patient's cells, such asfrom blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose.Deletions or insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabeled DNALigase III RNA or alternatively, radiolabeled DNA Ligase Ill antisenseDNA sequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

[0103] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242(1985)).

[0104] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase protection and S1 protectionor the chemical cleavage method (e.g., Cotton et al., PNAS, USA,85:4397-4401 (1985)).

[0105] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing, or the use of restriction enzymes, e.g.,restriction fragment length polymorphisms, and Southern blotting ofgenomic DNA. Also, mutations may be detected by in situ analysis.

[0106] In addition, some diseases are a result of, or are characterizedby, changes in gene expression which can be detected by changes in themRNA. Alternatively, the DNA Ligase III gene can be used as a referenceto identify individuals expressing a decreased level of DNA Ligase IIIprotein, e.g., by Northern blotting.

[0107] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0108] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0109] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0110] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For example, 2,000 bp is good, 4,000 isbetter, and more than 4,000 is probably not necessary to get goodresults a reasonable percentage of the time. For a review of thistechnique, see Verma et al., Human Chromosomes: a Manual of BasicTechniques, Pergamon Press, New York (1988).

[0111] Detailed analysis of 19 individual chromosomes using acombination of fractional length measurements and fluorescent bindingcombined with high-resolution image analysis indicated that Human DNALigase III is located within bands 17q11.2-12.

[0112] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes). The gene of the present invention has beenmapped to chromosome 13q33-34.

[0113] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0114] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0115] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0116] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0117] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0118] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0119] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0120] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0121] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0122] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0123] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0124] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0125] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0126] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLE 1

[0127] Bacterial Expression and Purification of DNA Ligase III

[0128] The DNA sequence encoding DNA Ligase III, ATCC # 97052, isinitially amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ end sequences of the processed DNA Ligase III gene. The 5′oligonucleotide primer has the sequence 5′ CGCGGATCCATGGCTGAGCAACGGTTCTG3′ (SEQ ID NO:3) contains a Bam HI restriction enzyme site (underlined)followed by 20 nucleotides of DNA Ligase III coding sequence startingfrom the presumed terminal amino acid of the processed protein codon.The 3′ sequence 5′ GCGTCTAGACTAGCAGGGAGCTACCAG 3′ (SEQ ID NO:4) containscomplementary sequences to a XbaI site (underlined) and is followed by18 nucleotides of DNA Ligase III at C-terminal of DNA Ligase III. Therestriction enzyme sites correspond to the restriction enzyme sites onthe bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, Calif.).pQE-9 encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 is then digested with Bam HI and Pst I. The amplified sequencesare ligated into pQE-9 and inserted in frame with the sequence encodingfor the histidine tag and the RBS. The ligation mixture is then used totransform E. coli strain M15/rep 4 (Qiagen, Inc.) under the trademarkM15/rep 4 by the procedure described in Sambrook, J. et al., MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989).M15/rep4 contains multiple copies of the plasmid pREP4, which expressesthe lacI repressor and also confers kanamycin resistance (Kan^(r)).Transformants are identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis. Clones containing thedesired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedprotein extract is purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984)) and eluted from the column in 6 molar guanidine HClpH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidineHCl, 100 mM sodium phosphate, 10 mmolar glutathione (reduced) and 2mmolar glutathione (oxidized). After incubation in this solution for 12hours the protein is dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 2

[0129] Cloning and Expression of DNA Ligase III Using the BaculovirusExpression System

[0130] A DNA sequence encoding full length DNA Ligase III protein, ATCC# 97052, is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene:

[0131] The 5′ primer has the sequence 5′ CGCGAATCCATGGCTGAGCAACGGTTCTG3′ (SEQ ID NO:5) and contains a BamHI restriction enzyme site (in bold)followed first by 20 nucleotides of N-terminal sequence (the initiationcodon for translation “ATG” is underlined).

[0132] The 3′ primer has the sequence 5′ GCGTCTAGACTAGCAGGGAGCTACCAG 3′(SEQ ID NO:6) and contains the cleavage site for the restrictionendonuclease XbaI (in bold) and 18 nucleotides complementary to theC-terminal sequence of the DNA Ligase III gene. The amplified sequenceswere isolated from a 1% agarose gel using a commercially available kit(“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment was thendigested with the endonucleases BamHI and XbaI and then purified againon a 1% agarose gel. This fragment is designated F2.

[0133] The vector pA2 (modification of pVL941 vector, discussed below)is used for the expression of the DNA Ligase III protein using thebaculovirus expression system (for review see: Summers, M. D. and Smith,G. E. 1987, A manual of methods for baculovirus vectors and insect cellculture procedures, Texas Agricultural Experimental Station Bulletin No.1555). This expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhidrosis virus (AcMNPV) followedby the recognition sites for the restriction endonucleases BamHI andXbaI. The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

[0134] The plasmid is digested with the restriction enzymes BamHI andXbaI and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The DNA is then isolated from a 1% agarosegel using the commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Calif.). This vector DNA is designated V2.

[0135] Fragment F2 and the dephosphorylated plasmid V2 were ligated withT4 DNA ligase. E. coli HB101 cells are then transformed and bacteriaidentified that contained the plasmid (pBac DNA Ligase III) with the DNALigase III gene using the enzymes BamHI and XbaI. The sequence of thecloned fragment is confirmed by DNA sequencing.

[0136] 5 μg of the plasmid pBac DNA Ligase III was co-transfected with1.0 μg of a commercially available linearized baculovirus (“BaculoGoldbaculovirus DNA”, Pharmingen, San Diego, Calif.) using the lipofectionmethod (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

[0137] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac DNALigase III are mixed in a sterile well of a microtiter plate containing50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to the Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivationcontinued at 27° C. for four days.

[0138] After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0139] Four days after the serial dilution, the viruses are added to thecells and blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

[0140] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-DNA Ligase III at a multiplicity of infection (MOI) of 2.Six hours later the medium is removed and replaced with SF900 III mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham)are added. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labeled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 3

[0141] Expression of Recombinant DNA Ligase III in COS Cells

[0142] The expression of plasmid, DNA Ligase III HA is derived from avector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin ofreplication, 2) ampicillin resistance gene, 3) E. coli replicationorgin, 4) CMV promoter followed by a polylinker region, a SV40 intronand polyadenylation site. A DNA fragment encoding the entire DNA LigaseIII precursor and a HA tag fused in frame to its 3′ end was cloned intothe polylinker region of the vector, therefore, the recombinant proteinexpression is directed under the CMV promoter. The HA tag correspond toan epitope derived from the influenza hemagglutinin protein aspreviously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M.Connolly, and R. Lerner, Cell 37:767 (1984)). The infusion of HA tag tothe target protein allows detection of the recombinant protein with anantibody that recognizes the HA epitope.

[0143] The plasmid construction strategy is described as follows:

[0144] The DNA sequence encoding DNA Ligase II, ATCC # 97052, isconstructed by PCR using two primers: the 5′ primer 5′CGCGAATCCATGGCTGAGCAACGGTTCTG 3′ (SEQ ID NO:7) contains an BamHI site(underlined) followed by 20 nucleotides of DNA Ligase III codingsequence starting from the initiation codon; the 3′ sequence 5′GCGTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAGCAGGGAGCTACCA GTC 3′ (SEQ IDNO:8) contains complementary sequences to an XbaI site (underlined),translation stop codon, HA tag and the last 17 nucleotides of the DNALigase III coding sequence (not including the stop codon). Therefore,the PCR product contains an BamHI site, DNA Ligase III coding sequencefollowed by HA tag fused in frame, a translation termination stop codonnext to the HA tag, and an XbaI site. The PCR amplified DNA fragment andthe vector, pcDNAI/Amp, are digested with BamHI and XbaI restrictionenzyme and ligated. The ligation mixture is transformed into E. colistrain SURE (Stratagene Cloning Systems, La Jolla, Calif.) thetransformed culture is plated on ampicillin media plates and resistantcolonies are selected. Plasmid DNA is isolated from transformants andexamined by restriction analysis for the presence of the correctfragment. For expression of the recombinant DNA Ligase III, COS cellsare transfected with the expression vector by DEAE-DEXTRAN method (J.Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989)). The expression of the DNALigase III HA protein is detected by radiolabeling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labeledfor 8 hours with ³⁵S-cysteine two days post transfection. Culture mediaare then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I.et al., Id. 37:767 (1984)). Both cell lysate and culture media areprecipitated with a HA specific monoclonal antibody. Proteinsprecipitated are analyzed on 15% SDS-PAGE gels.

EXAMPLE 4

[0145] Expression Pattern of DNA Ligase III in Human Tissue

[0146] Northern blot analysis may be performed to examine the levels ofexpression of DNA Ligase III in human tissues. Total cellular RNAsamples are isolated with RNAzol™ B system (Biotecx Laboratories, Inc.Houston, Tex.) About 15 μg of total RNA isolated from each human tissuespecified is separated on 1% agarose gel and blotted onto a nylon filter(Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring HarborPress, (1989)). The labeling reaction is done according to theStratagene Prime-It kit with 50 ng DNA fragment. The labeled DNA ispurified with a Select-G-50 column (5 Prime-3 Prime, Inc. Boulder,Colo.). The filter containing the particular RNA blot is then hybridizedwith radioactive labeled full length DNA Ligase III gene at 1,000,000cpm/ml in 0.5 M NaPO₄, pH 7.4 and 7% SDS overnight at 65° C. After washtwice at room temperature and twice at 60° C. with 0.5×SSC, 0.1% SDS,the filter is then exposed at −70° C. overnight with an intensifyingscreen. The message RNA for DNA Ligase III is abundant in the testis,prostate, heart, and thymus.

EXAMPLE 5

[0147] In Vitro Transcription/Translation of cDNA Clones

[0148] Putative full-length CDNA clone was subcloned as follows: DNAligase III was subcloned as a Sal I/Not I restriction fragment into themultiple cloning sire of pSPORT (Life Technologies), with the 5′ endproximal to the T7 promoter; the DNA ligase III plasmid constructs (1μg) was linearized with either Not I or Xho I (New England Biolabs),downstream of the cDNA insert, then transcribed and capped at 36° C. for30 minutes with T7 polymerase and the mCAP RNA capping kit (Stratagene).The reactions were terminated by incubation with 10 units RNase-freeDNase at 37° C. for 5 minutes. Following phenol/chloroform extractionand ethanol precipitation, the in vitro transcription products wereresuspended in 20 μl 10 mM Tris-HCl/1 mM EDTA, pH 8.0 (TE). Thetranscript (0 to 5 μl, made up to a final volume of 5 μl with water) wastranslated in 20 μl rabbit reticulocyte lysate (Amersham) at 30° C. for90 minutes. In order to radiolabel the product of in vitro translation,reaction was supplemented with 20 μCi [³⁵S]methionine (3000 Ci mmol⁻¹,Amersham). Translations were terminated by incubation with 5 μl of 400ml⁻¹ RNase A/50 mM EDTA at 37° C. for 15 minutes (30 μl final volume).Samples (5 μl) of translations carried out in the presence of[³⁵S]methionine were analyzed by electrophoresis in SDS-7.5%polyacrylamide gels and autoradiography. Non-radiolabeled translationproducts were assayed for ability to form protein-adenylate complexesafter removal of ATP by chromatography through spun 1 ml columns ofSephadex G50 (Pharmacia) equilibrated with TE.

EXAMPLE 6

[0149] DNA Ligase Assays

[0150] 5 μl samples from in vitro translations were adenylylated inreaction mixtures (30 μl) containing 60 mM Tris HCl (pH 8.0), 10 mMMgCl₂, 50 μg ml⁻¹ BSA, 5 mM DTT and 1 μCi [α-³²P] ATP (3000 Ci mmol⁻¹,Amersham) at 20° C. for 10 minutes and then analyzed by electrophoresisin SDS-7.5% polyacrylamide gels and autoradiography. In order to monitortransfer of [³²P]AMP from protein-adenylate to a nicked DNA substrate, 5μl samples from adenylylation reactions were incubated for further timeperiods with or without the addition of 500 ng non-radiolabeledoligo(dT)₁₆-poly(dA), as described previously. The ability to transfer[³²P]AMP from enzyme-adenylate to the hybrid substrates,oligo(dT)-poly(rA) or oligo(rA)-poly(dT), differentiates DNA ligase I,II and III. However, both these latter substrates were rapidly degradedby an RNase H activity upon incubation in the reticulocyte lysate, evenwhen mixtures were used directly without termination of translationreactions by addition of RNase A.

EXAMPLE 7

[0151] Expression of DNA Ligase III via Gene Therapy

[0152] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0153] Moloney murine leukemia virus is digested and treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads. The DNA Ligase III cDNA (see FIGS.1A-1J) is isolated and the ends of this fragment are treated with DNApolymerase in order to fill in the recessed ends and create blunt ends.

[0154] Equal quantities of the Moloney murine leukemia virus linearbackbone and the gene are added together, in the presence of T4 DNAligase. The resulting mixture is maintained under conditions appropriatefor ligation of the two fragments. The ligation mixture was used totransform bacteria HB101, which were then plated onto agar-containingkanamycin for the purpose of confirming that the vector had the DNALigase III gene properly inserted.

[0155] PE501 packaging cells are grown in tissue culture to confluentdensity in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum(CS), penicillin and streptomycin. The Moloney murine leukemia virusvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the DNA Ligase III gene.

[0156] Fresh media is added to the transduced producer cells, andsubsequently the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells.

[0157] The engineered fibroblasts are then injected into the into ahost, for example, a rat, either alone or after having been grown toconfluence on cytodex 3 microcarrier beads. The fibroblasts now producethe protein product and the biological actions of DNA Ligase III areconveyed to the host.

[0158] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 11 1 3417 DNA Homo sapiens CDS (334)..(3102) 1 ccacgcgtcc ggcagcctgtatgagcaagt gccgaggcct acggtgagcg ccggagccgg 60 agaggcagct atatgtctttggctttcaag atcttctttc cacaaaccct ccgtgcactc 120 agccgaaaag aactgtgcctattccgaaaa catcactggc gtgatgtaag acaattcagc 180 cagtggtcag aaacagatctgcttcatgga catcccctct tcctgagaag aaagcctgtt 240 ctatcattcc agggaagccatctaagatca cgtgccacct accttgtttt cttgccaggg 300 ttgcatgtgg gactctgcagtggcccctgt gag atg gct gag caa cgg ttc tgt 354 Met Ala Glu Gln Arg PheCys 1 5 gtg gac tat gcc aag cgt ggc aca gct ggc tgc aaa aaa tgc aag gaa402 Val Asp Tyr Ala Lys Arg Gly Thr Ala Gly Cys Lys Lys Cys Lys Glu 1015 20 aag att gtg aag ggc gta tgc cga att ggc aaa gtg gtg ccc aat ccc450 Lys Ile Val Lys Gly Val Cys Arg Ile Gly Lys Val Val Pro Asn Pro 2530 35 ttc tca gag tct ggg ggt gat atg aaa gag tgg tac cac att aaa tgc498 Phe Ser Glu Ser Gly Gly Asp Met Lys Glu Trp Tyr His Ile Lys Cys 4045 50 55 atg ttt gag aaa cta gag cgg gcc cgg gcc acc aca aaa aaa atc gag546 Met Phe Glu Lys Leu Glu Arg Ala Arg Ala Thr Thr Lys Lys Ile Glu 6065 70 gac ctc aca gag ctg gaa ggc tgg gaa gag ctg gaa gat aat gag aag594 Asp Leu Thr Glu Leu Glu Gly Trp Glu Glu Leu Glu Asp Asn Glu Lys 7580 85 gaa cag ata acc cag cac att gca gat ctg tct tct aag gca gca ggt642 Glu Gln Ile Thr Gln His Ile Ala Asp Leu Ser Ser Lys Ala Ala Gly 9095 100 aca cca aag aag aaa gct gtt gtc cag gct aag ttg aca acc act ggc690 Thr Pro Lys Lys Lys Ala Val Val Gln Ala Lys Leu Thr Thr Thr Gly 105110 115 cag gtg act tct cca gtg aaa ggc gcc tca ttt gtc acc agt acc aat738 Gln Val Thr Ser Pro Val Lys Gly Ala Ser Phe Val Thr Ser Thr Asn 120125 130 135 ccc cgg aaa ttt tct ggc ttt tca gcc aag ccc aac aac tct ggggaa 786 Pro Arg Lys Phe Ser Gly Phe Ser Ala Lys Pro Asn Asn Ser Gly Glu140 145 150 gcc ccc tcg agc ccc acc cct aag aga agt ctg tct tca agc aaatgt 834 Ala Pro Ser Ser Pro Thr Pro Lys Arg Ser Leu Ser Ser Ser Lys Cys155 160 165 gac ccc agg cat aag gac tgt ctg cta cgg gag ttt cga aag ttatgc 882 Asp Pro Arg His Lys Asp Cys Leu Leu Arg Glu Phe Arg Lys Leu Cys170 175 180 gcc atg gtg gcc gat aat cct agc tac aac acg aag acc cag atcatc 930 Ala Met Val Ala Asp Asn Pro Ser Tyr Asn Thr Lys Thr Gln Ile Ile185 190 195 cag gac ttc ctt cgg aaa ggc tca gca gga gat ggt ttc cac ggtgat 978 Gln Asp Phe Leu Arg Lys Gly Ser Ala Gly Asp Gly Phe His Gly Asp200 205 210 215 gtg tac cta aca gtg aag ctg ctg ctg cca gga gtc att aagact gtt 1026 Val Tyr Leu Thr Val Lys Leu Leu Leu Pro Gly Val Ile Lys ThrVal 220 225 230 tac aac ttg aac gat aag cag att gtg aag ctt ttc agt cgcatt ttt 1074 Tyr Asn Leu Asn Asp Lys Gln Ile Val Lys Leu Phe Ser Arg IlePhe 235 240 245 aac tgc aac cca gat gat atg gca cgg gac cta gag cag ggtgac gtg 1122 Asn Cys Asn Pro Asp Asp Met Ala Arg Asp Leu Glu Gln Gly AspVal 250 255 260 tca gag aca atc aga gtc ttc ttt gag cag agc aag tct ttcccc cca 1170 Ser Glu Thr Ile Arg Val Phe Phe Glu Gln Ser Lys Ser Phe ProPro 265 270 275 gct gcc aag agc ctc ctt acc atc cag gaa gtg gat gag ttcctt ctg 1218 Ala Ala Lys Ser Leu Leu Thr Ile Gln Glu Val Asp Glu Phe LeuLeu 280 285 290 295 cgg ctg tcc aag ctc acc aag gag gat gag cag caa caggcc cta cag 1266 Arg Leu Ser Lys Leu Thr Lys Glu Asp Glu Gln Gln Gln AlaLeu Gln 300 305 310 gac att gcc tcc agg tgt aca gcc aat gac ctt aaa tgcatc atc agg 1314 Asp Ile Ala Ser Arg Cys Thr Ala Asn Asp Leu Lys Cys IleIle Arg 315 320 325 ttg atc aaa cat gat ctg aag atg aac tca ggt gca aaacat gtg tta 1362 Leu Ile Lys His Asp Leu Lys Met Asn Ser Gly Ala Lys HisVal Leu 330 335 340 gac gcc ctt gac ccc aat gcc tat gaa gcc ttc aaa gcctcg cgc aac 1410 Asp Ala Leu Asp Pro Asn Ala Tyr Glu Ala Phe Lys Ala SerArg Asn 345 350 355 ctg cag gat gtg gtg gag cgg gtc ctt cac aac gcg caggag gtg gag 1458 Leu Gln Asp Val Val Glu Arg Val Leu His Asn Ala Gln GluVal Glu 360 365 370 375 aag gag ccg ggc cag aga cga gct ctg agc gtc caggcc tcg ctg atg 1506 Lys Glu Pro Gly Gln Arg Arg Ala Leu Ser Val Gln AlaSer Leu Met 380 385 390 aca cct gtg cag ccc atg ttg gcg gag gcc tgc aagtcc gtt gag tat 1554 Thr Pro Val Gln Pro Met Leu Ala Glu Ala Cys Lys SerVal Glu Tyr 395 400 405 gca atg aag aaa tgt ccc aat ggc atg ttc tct gagatc aag tac gat 1602 Ala Met Lys Lys Cys Pro Asn Gly Met Phe Ser Glu IleLys Tyr Asp 410 415 420 gga gag cga gtc cag gtg cat aag aat gga gac cacttc agc tac ttc 1650 Gly Glu Arg Val Gln Val His Lys Asn Gly Asp His PheSer Tyr Phe 425 430 435 agc cgc agt ctc aag ccc gtc ctt cct cac aag gtggcc cac ttt aag 1698 Ser Arg Ser Leu Lys Pro Val Leu Pro His Lys Val AlaHis Phe Lys 440 445 450 455 gac tac att ccc cag gct ttt cct ggg ggc cacagc atg atc ttg gat 1746 Asp Tyr Ile Pro Gln Ala Phe Pro Gly Gly His SerMet Ile Leu Asp 460 465 470 tct gaa gtg ctt ctg att gac aac aag aca ggcaaa cca ctg ccc ttt 1794 Ser Glu Val Leu Leu Ile Asp Asn Lys Thr Gly LysPro Leu Pro Phe 475 480 485 ggg act ctg gga gta cac aag aaa gca gcc ttccag gat gct aat gtc 1842 Gly Thr Leu Gly Val His Lys Lys Ala Ala Phe GlnAsp Ala Asn Val 490 495 500 tgc ctg ttt gtt ttt gat tgt atc tac ttt aatgat gtc agc ttg atg 1890 Cys Leu Phe Val Phe Asp Cys Ile Tyr Phe Asn AspVal Ser Leu Met 505 510 515 gac aga cct ctg tgt gag cgg cgg aag ttt cttcat gac aac atg gtt 1938 Asp Arg Pro Leu Cys Glu Arg Arg Lys Phe Leu HisAsp Asn Met Val 520 525 530 535 gaa att cca aac cgg atc atg ttc tca gaaatg aag cga gtc aca aaa 1986 Glu Ile Pro Asn Arg Ile Met Phe Ser Glu MetLys Arg Val Thr Lys 540 545 550 gct ttg gac ttg gct gac atg ata acc cgggtg atc cag gag gga ttg 2034 Ala Leu Asp Leu Ala Asp Met Ile Thr Arg ValIle Gln Glu Gly Leu 555 560 565 gag ggg ctg gtg ctg aag gat gtg aag ggtaca tat gag cct ggg aag 2082 Glu Gly Leu Val Leu Lys Asp Val Lys Gly ThrTyr Glu Pro Gly Lys 570 575 580 cgg cac tgg ctg aaa gtg aag aaa gac tatttg aac gag ggg gcc atg 2130 Arg His Trp Leu Lys Val Lys Lys Asp Tyr LeuAsn Glu Gly Ala Met 585 590 595 gcc gac aca gct gac ctg gtg gtc ctt ggagcc ttc tat ggg caa ggg 2178 Ala Asp Thr Ala Asp Leu Val Val Leu Gly AlaPhe Tyr Gly Gln Gly 600 605 610 615 agc aaa ggc ggc atg atg tca atc ttcctc atg ggc tgc tac gac cct 2226 Ser Lys Gly Gly Met Met Ser Ile Phe LeuMet Gly Cys Tyr Asp Pro 620 625 630 ggc agc cag aag tgg tgc aca gtc accaag tgt gca gga ggc cat gat 2274 Gly Ser Gln Lys Trp Cys Thr Val Thr LysCys Ala Gly Gly His Asp 635 640 645 gat gcc acg ctt gcc cgc ctg cag aatgaa cta gac atg gtg aag atc 2322 Asp Ala Thr Leu Ala Arg Leu Gln Asn GluLeu Asp Met Val Lys Ile 650 655 660 agc aag gac ccc agc aaa ata ccc agctgg ttg aag gtc aac aag atc 2370 Ser Lys Asp Pro Ser Lys Ile Pro Ser TrpLeu Lys Val Asn Lys Ile 665 670 675 tac tat cct gac ttc atc gtc cca gaccca aag aaa gct gcc gtg tgg 2418 Tyr Tyr Pro Asp Phe Ile Val Pro Asp ProLys Lys Ala Ala Val Trp 680 685 690 695 gag atc aca ggg gct gaa ttc tccaaa tcg gag gct cat aca gct gac 2466 Glu Ile Thr Gly Ala Glu Phe Ser LysSer Glu Ala His Thr Ala Asp 700 705 710 ggg atc tcc atc cga ttc cct cgctgc acc cga atc cga gat gat aag 2514 Gly Ile Ser Ile Arg Phe Pro Arg CysThr Arg Ile Arg Asp Asp Lys 715 720 725 gac tgg aaa tct gcc act aac cttccc caa ctc aag gaa ctg tac cag 2562 Asp Trp Lys Ser Ala Thr Asn Leu ProGln Leu Lys Glu Leu Tyr Gln 730 735 740 ttg tcc aag gag aag gca gac ttcact gta gtg gct gga gat gag ggg 2610 Leu Ser Lys Glu Lys Ala Asp Phe ThrVal Val Ala Gly Asp Glu Gly 745 750 755 agc tcc act aca ggg ggt agc agtgaa gag aat aag ggt ccc tca ggg 2658 Ser Ser Thr Thr Gly Gly Ser Ser GluGlu Asn Lys Gly Pro Ser Gly 760 765 770 775 tct gct gtg tcc cgc aag gccccc agc aag ccc tca gcc agt acc aag 2706 Ser Ala Val Ser Arg Lys Ala ProSer Lys Pro Ser Ala Ser Thr Lys 780 785 790 aaa gca gaa ggg aag ctg agtaac tcc aac agc aaa gat ggc aac atg 2754 Lys Ala Glu Gly Lys Leu Ser AsnSer Asn Ser Lys Asp Gly Asn Met 795 800 805 cag act gca aag cct tcc gctatg aag gtg ggg gag aag ctg gcc aca 2802 Gln Thr Ala Lys Pro Ser Ala MetLys Val Gly Glu Lys Leu Ala Thr 810 815 820 aag tct tct cca gtg aaa gtaggg gag aag cgg aaa gct gct gat gag 2850 Lys Ser Ser Pro Val Lys Val GlyGlu Lys Arg Lys Ala Ala Asp Glu 825 830 835 acg ctg tgc caa aca aag gtattg ctg gac atc ttc act ggg gtg cgg 2898 Thr Leu Cys Gln Thr Lys Val LeuLeu Asp Ile Phe Thr Gly Val Arg 840 845 850 855 ctt tac ttg cca ccc tccaca cca gac ttc agc cgt ctc aga cgc tac 2946 Leu Tyr Leu Pro Pro Ser ThrPro Asp Phe Ser Arg Leu Arg Arg Tyr 860 865 870 ttt gtg gca ttc gac ggggac ctg gta cag gaa ttt gat atg act tca 2994 Phe Val Ala Phe Asp Gly AspLeu Val Gln Glu Phe Asp Met Thr Ser 875 880 885 gcc acg cac gtg ctg ggtagc agg gac aag aac cct gcg gcc cag cag 3042 Ala Thr His Val Leu Gly SerArg Asp Lys Asn Pro Ala Ala Gln Gln 890 895 900 gtc tcc cca gag tgg atttgg gca tgt atc cgg aaa cgg aga ctg gta 3090 Val Ser Pro Glu Trp Ile TrpAla Cys Ile Arg Lys Arg Arg Leu Val 905 910 915 gct ccc tgc taggtttgctgtc ttccctctcc ctcaggccat actctccttt 3142 Ala Pro Cys 920accatactat tggactggac tcaggctgga ggcagataga cacagtatag ggggaatggg 3202cttgcttctc ccaaacccac cagttctcca ctgtctcttc tggaccagga attagttgct 3262gtgggtgcca cagctgaagt cagtttgtct tgctggttta aatagatctt tcagagctgg 3322gtgctgggtt tgccatcttt ttgttttctt tgaaaagcag cttagttacc ctttttataa 3382ataaaatatc ttgcagttaa aaaaaaaaaa aaaaa 3417 2 922 PRT Homo sapiensACT_SITE (421)..(421) Active site lysine 2 Met Ala Glu Gln Arg Phe CysVal Asp Tyr Ala Lys Arg Gly Thr Ala 1 5 10 15 Gly Cys Lys Lys Cys LysGlu Lys Ile Val Lys Gly Val Cys Arg Ile 20 25 30 Gly Lys Val Val Pro AsnPro Phe Ser Glu Ser Gly Gly Asp Met Lys 35 40 45 Glu Trp Tyr His Ile LysCys Met Phe Glu Lys Leu Glu Arg Ala Arg 50 55 60 Ala Thr Thr Lys Lys IleGlu Asp Leu Thr Glu Leu Glu Gly Trp Glu 65 70 75 80 Glu Leu Glu Asp AsnGlu Lys Glu Gln Ile Thr Gln His Ile Ala Asp 85 90 95 Leu Ser Ser Lys AlaAla Gly Thr Pro Lys Lys Lys Ala Val Val Gln 100 105 110 Ala Lys Leu ThrThr Thr Gly Gln Val Thr Ser Pro Val Lys Gly Ala 115 120 125 Ser Phe ValThr Ser Thr Asn Pro Arg Lys Phe Ser Gly Phe Ser Ala 130 135 140 Lys ProAsn Asn Ser Gly Glu Ala Pro Ser Ser Pro Thr Pro Lys Arg 145 150 155 160Ser Leu Ser Ser Ser Lys Cys Asp Pro Arg His Lys Asp Cys Leu Leu 165 170175 Arg Glu Phe Arg Lys Leu Cys Ala Met Val Ala Asp Asn Pro Ser Tyr 180185 190 Asn Thr Lys Thr Gln Ile Ile Gln Asp Phe Leu Arg Lys Gly Ser Ala195 200 205 Gly Asp Gly Phe His Gly Asp Val Tyr Leu Thr Val Lys Leu LeuLeu 210 215 220 Pro Gly Val Ile Lys Thr Val Tyr Asn Leu Asn Asp Lys GlnIle Val 225 230 235 240 Lys Leu Phe Ser Arg Ile Phe Asn Cys Asn Pro AspAsp Met Ala Arg 245 250 255 Asp Leu Glu Gln Gly Asp Val Ser Glu Thr IleArg Val Phe Phe Glu 260 265 270 Gln Ser Lys Ser Phe Pro Pro Ala Ala LysSer Leu Leu Thr Ile Gln 275 280 285 Glu Val Asp Glu Phe Leu Leu Arg LeuSer Lys Leu Thr Lys Glu Asp 290 295 300 Glu Gln Gln Gln Ala Leu Gln AspIle Ala Ser Arg Cys Thr Ala Asn 305 310 315 320 Asp Leu Lys Cys Ile IleArg Leu Ile Lys His Asp Leu Lys Met Asn 325 330 335 Ser Gly Ala Lys HisVal Leu Asp Ala Leu Asp Pro Asn Ala Tyr Glu 340 345 350 Ala Phe Lys AlaSer Arg Asn Leu Gln Asp Val Val Glu Arg Val Leu 355 360 365 His Asn AlaGln Glu Val Glu Lys Glu Pro Gly Gln Arg Arg Ala Leu 370 375 380 Ser ValGln Ala Ser Leu Met Thr Pro Val Gln Pro Met Leu Ala Glu 385 390 395 400Ala Cys Lys Ser Val Glu Tyr Ala Met Lys Lys Cys Pro Asn Gly Met 405 410415 Phe Ser Glu Ile Lys Tyr Asp Gly Glu Arg Val Gln Val His Lys Asn 420425 430 Gly Asp His Phe Ser Tyr Phe Ser Arg Ser Leu Lys Pro Val Leu Pro435 440 445 His Lys Val Ala His Phe Lys Asp Tyr Ile Pro Gln Ala Phe ProGly 450 455 460 Gly His Ser Met Ile Leu Asp Ser Glu Val Leu Leu Ile AspAsn Lys 465 470 475 480 Thr Gly Lys Pro Leu Pro Phe Gly Thr Leu Gly ValHis Lys Lys Ala 485 490 495 Ala Phe Gln Asp Ala Asn Val Cys Leu Phe ValPhe Asp Cys Ile Tyr 500 505 510 Phe Asn Asp Val Ser Leu Met Asp Arg ProLeu Cys Glu Arg Arg Lys 515 520 525 Phe Leu His Asp Asn Met Val Glu IlePro Asn Arg Ile Met Phe Ser 530 535 540 Glu Met Lys Arg Val Thr Lys AlaLeu Asp Leu Ala Asp Met Ile Thr 545 550 555 560 Arg Val Ile Gln Glu GlyLeu Glu Gly Leu Val Leu Lys Asp Val Lys 565 570 575 Gly Thr Tyr Glu ProGly Lys Arg His Trp Leu Lys Val Lys Lys Asp 580 585 590 Tyr Leu Asn GluGly Ala Met Ala Asp Thr Ala Asp Leu Val Val Leu 595 600 605 Gly Ala PheTyr Gly Gln Gly Ser Lys Gly Gly Met Met Ser Ile Phe 610 615 620 Leu MetGly Cys Tyr Asp Pro Gly Ser Gln Lys Trp Cys Thr Val Thr 625 630 635 640Lys Cys Ala Gly Gly His Asp Asp Ala Thr Leu Ala Arg Leu Gln Asn 645 650655 Glu Leu Asp Met Val Lys Ile Ser Lys Asp Pro Ser Lys Ile Pro Ser 660665 670 Trp Leu Lys Val Asn Lys Ile Tyr Tyr Pro Asp Phe Ile Val Pro Asp675 680 685 Pro Lys Lys Ala Ala Val Trp Glu Ile Thr Gly Ala Glu Phe SerLys 690 695 700 Ser Glu Ala His Thr Ala Asp Gly Ile Ser Ile Arg Phe ProArg Cys 705 710 715 720 Thr Arg Ile Arg Asp Asp Lys Asp Trp Lys Ser AlaThr Asn Leu Pro 725 730 735 Gln Leu Lys Glu Leu Tyr Gln Leu Ser Lys GluLys Ala Asp Phe Thr 740 745 750 Val Val Ala Gly Asp Glu Gly Ser Ser ThrThr Gly Gly Ser Ser Glu 755 760 765 Glu Asn Lys Gly Pro Ser Gly Ser AlaVal Ser Arg Lys Ala Pro Ser 770 775 780 Lys Pro Ser Ala Ser Thr Lys LysAla Glu Gly Lys Leu Ser Asn Ser 785 790 795 800 Asn Ser Lys Asp Gly AsnMet Gln Thr Ala Lys Pro Ser Ala Met Lys 805 810 815 Val Gly Glu Lys LeuAla Thr Lys Ser Ser Pro Val Lys Val Gly Glu 820 825 830 Lys Arg Lys AlaAla Asp Glu Thr Leu Cys Gln Thr Lys Val Leu Leu 835 840 845 Asp Ile PheThr Gly Val Arg Leu Tyr Leu Pro Pro Ser Thr Pro Asp 850 855 860 Phe SerArg Leu Arg Arg Tyr Phe Val Ala Phe Asp Gly Asp Leu Val 865 870 875 880Gln Glu Phe Asp Met Thr Ser Ala Thr His Val Leu Gly Ser Arg Asp 885 890895 Lys Asn Pro Ala Ala Gln Gln Val Ser Pro Glu Trp Ile Trp Ala Cys 900905 910 Ile Arg Lys Arg Arg Leu Val Ala Pro Cys 915 920 3 29 DNAArtificial Sequence Contains a Bam HI restriction enzyme site 3cgcggatcca tggctgagca acggttctg 29 4 27 DNA Artificial Sequence Containscomplementary sequences to a XbaI site 4 gcgtctagac tagcagggag ctaccag27 5 29 DNA Artificial Sequence Contains a BamHI restriction enzyme site5 cgcgaatcca tggctgagca acggttctg 29 6 29 DNA Artificial SequenceContains the cleavage site for the restriction endonuclease XbaI 6cgcgaatcca tggctgagca acggttctg 29 7 29 DNA Artificial Sequence Containsa BamHI site 7 cgcgaatcca tggctgagca acggttctg 29 8 56 DNA ArtificialSequence Contains complementary sequences to an XbaI site (underlined),translation stop codon, and an HA tag 8 gcgtctagat caagcgtagt ctgggacgtcgtatgggtag cagggagcta ccagtc 56 9 21 PRT Homo sapiens 9 Lys Cys Pro AsnGly Met Phe Ser Glu Ile Lys Tyr Asp Gly Glu Arg 1 5 10 15 Val Gln ValHis Lys 20 10 8 PRT Homo sapiens MISC_FEATURE (1)..(1) Xaa is any aminoacid 10 Xaa Lys Xaa Asp Gly Xaa Arg Xaa 1 5 11 8 PRT Homo sapiensMISC_FEATURE (2)..(2) Xaa is any amino acid 11 Glu Xaa Lys Tyr Asp GlyXaa Arg 1 5

What is claimed is:
 1. A method of producing an antibody thatspecifically binds the polypeptide of SEQ ID NO:2 comprising: (a)introducing a polypeptide comprising at least 50 contiguous amino acidsof SEQ ID NO:2 into an animal; and (b) recovering said antibody.
 2. Themethod of claim 1 wherein the antibody binds a polypeptide consisting ofamino acids 2 to 922 of SEQ ID NO:2.
 3. The method of claim 1 whereinthe antibody is a polyclonal antibody.
 4. The method of claim 1 thatalso comprises the step of generating a hybridoma prior to recoveringsaid antibody.
 5. The method of claim 4 wherein the antibody is amonoclonal antibody.
 6. A method of producing an antibody thatspecifically binds the full-length polypeptide encoded by the cDNA inATCC Deposit No. 97052 comprising: (a) introducing a polypeptidecomprising at least 50 contiguous amino acids encoded by ATCC DepositNo. 97052 into an animal; and (b) recovering said antibody.
 7. Themethod of claim 6 wherein the antibody binds a polypeptide consisting ofthe full-length protein encoded by the cDNA contained in ATCC DepositNo. 97052, excepting the N-terminal methionine.
 8. The method of claim 6wherein the antibody is a polyclonal antibody.
 9. The method of claim 6that also comprises the step of generating a hybridoma prior torecovering said antibody.
 10. The method of claim 9 wherein the antibodyis a monoclonal antibody.
 11. A method of producing an antibody thatspecifically binds the polypeptide of SEQ ID NO:2 comprising: (a)screening a single chain or Fab expression library to identify anantibody that specifically binds a polypeptide comprising at least 50amino acids of SEQ ID NO:2; and (b) recovering said antibody from saidlibrary.
 12. The method of claim 11 wherein the antibody is a singlechain antibody.
 13. The method of claim 11 wherein the antibody is anFab fragment.
 14. The method of claim 11 wherein the polypeptidecomprising at least 50 amino acids of SEQ ID NO:2 consists of amino acidresidues 2 to 922 of SEQ ID NO:2.
 15. The method of claim 11 wherein thepolypeptide comprising at least 50 amino acids of SEQ ID NO:2 consistsof amino acid residues 1 to 922 of SEQ ID NO:2.
 16. A method ofproducing an antibody that specifically binds the polypeptide encoded bythe cDNA in ATCC Deposit No. 97052 comprising: (a) screening a singlechain or Fab expression library to identify an antibody that binds apolypeptide comprising at least 50 amino acids of the polypeptideencoded by the cDNA in ATCC Deposit No. 97052; and (b) recovering saidantibody from said library.
 17. The method of claim 16 wherein theantibody is a single chain antibody.
 18. The method of claim 16 whereinthe antibody is an Fab fragment.
 19. The method of claim 16 wherein thepolypeptide comprising at least 50 amino acids of the polypeptideencoded by the human cDNA in ATCC Deposit No. 97052 consists of thefull-length polypeptide encoded by the cDNA in ATCC Deposit No. 97052,excepting the N-terminal methionine.
 20. The method of claim 16 whereinthe polypeptide comprising at least 50 amino acids of the polypeptideencoded by the human cDNA in ATCC Deposit No. 97052 consists of thefull-length polypeptide encoded by the cDNA in ATCC Deposit No. 97052.